Work on this project has focused on the achondroplasia family of skeletal dysplasias, an autosomal recessive form of skeletal dysplasia, Cartilage Hair Hypoplasia (CHH), and the Schwarz-Jampel syndrome. The achondroplasia family of skeletal dysplasias includes three previously recognized diagnoses and one that has been defined under this project. The three well-established conditions are achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Work published by our lab in the past described a new syndrome, Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN). All four disorders in this family of conditions are caused by mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3). Cartilage hair hypoplasia has recently been found to be caused by mutations in the RNMP gene, which encodes an RNA that participates in the mitochondrial Ribonuclear Protein complex. Mutational analysis in Amish patients with CHH and non-Amish CHH patients is ongoing. Our collaborative work with Dr. Yoshi Yamada and his colleagues at the NINDR has demonstrated that the Schwarz-Jampel syndrome is caused by mutations in the perlecan gene. Work over the past two years has focused on the creation and characterization of two mouse models for FGFR3 disorders. Using a knock-in strategy, mice heterozygous for K644E and K644M mutations have been generated. Ongoing work is focused on creation of mice with K644N mutations, to model the human disorder hypochondroplasia. We are anticipating functional genomic and proteomic analysis of the nervous system and skeleton of these mice, in order to better understand the pathogenesis of these three disorders. Over the coming year, Dr. Shepherd Schurman will be working to create a mouse model for the skeletal dysplasia Cartilage Hair Hypoplasia. Dr. ZJ Zhang has been using in vitro methods to create and enhance an organ culture method for the development of cartilage. The proximal and distal parts of sterna of chick embryos represent cartilage undergoing endochondral ossification and hyaline cartilage respectively. Cartilage explants from both regions were exposed to pulsed low-intensity ultrasound (PLIUS). An anabolic effect of PLIUS on matrix production was shown by an increase of up to 10-20% in quantitative immunohistochemical staining for type II collagen and aggrecan in the two parts of the sternum. PLIUS also increased type X collagen staining by up to 10% in certain regions of the proximal part of the sternum. Staining for type X collagen was negative in the distal part of the sternum in both PLIUS and control groups. These results suggest that PLIUS may stimulate bone formation by increasing hypertrophy of chondrocytes directed to terminal differentiation. However, PLIUS did not induce hypertrophy in hyaline cartilage; moreover, increased matrix synthesis indicates a potential role in cartilage repair. In studies designed to investigate the utility of pellet-culture for cartilage engineering, isolated chondrocytes were grown in pellet culture for 2 weeks. The chondrocyte phenotype was stabilized in this specially designed culture system as show by aggrecan staining and immunohistochemistry for type II collagen. During the culture period, chondrocytes maintained expression of type IX collagen, which functions to link collagen fiber with aggrecan. The reconstruction of extracellular matrix was demonstrated by electron microscopy. By 2 weeks, the fiber network in the engineered cartilage was similar to the native cartilage at fiber diameter and density, but the fiber orientation was less organized. One peer-reviewed publication is in press as a result of Dr. Zhang's work: Zhang Z., Huckle J., Francomano C., Spencer R. The influences of pulsed low-intensity ultrasound on the matrix productions of chondrocytes at different differentiation stages. Ultrasound Med Biol (in press). An abstract will be presented at the International Cartilage Repair Society Symposium in Toronto in October 2002: Zhang Z., Huckle J., Francomano C., Spencer R. The anabolic effect of pulsed low-intensity ultrasound on hyaline cartilage. The 4th International Cartilage Repair Society Symposium, Toronto, 2002 The HGIMS is also continuing to maintain the Skeletal Gene Database, a publically available electronic resource cataloguing all genes and expressed sequences found in skeletal tissue, and all known associations between genes and disease states or traits affecting the skeletal system. Clinical efforts include examination of the causes of morbidity and mortality in achondroplasia, and description of previously uncharacterized skeletal dysplasias.